1. Field of the Invention
The field of the present invention relates to the transplanting of organs and tissues, and more particularly to the production in a surrogate of regulatory cells and factors capable of generating immune tolerance to a graft organ in the recipient and to subsequently transplanting xenografts from the surrogate to a recipient. The invention also relates to methods for producing within a surrogate, organs for transplant that are repopulated with cells from the organ graft recipient, lessening the antigen difference and therefore the risk of rejection.
2. Review Of The Related Art
The normal immune system is capable of specifically differentiating between "self" and foreign entities, with foreign entities including infectious agents. The ability to differentiate self from foreign entities is established naturally during fetal development, when the developing immune system of the fetus is programmed to recognize presented antigens as self; i.e. as antigens of the fetus. Several mechanisms are responsible for immune tolerance; including suppression, negative selection, and anergy. Suppression refers to the inhibition of lymphocytes that are reactive to self antigens. Negative selection refers to prevention of the development of immune clones capable of reacting with self antigens. Anergy refers to cells that recognize self but fail to proliferate or function in response to the self antigen.
Suppressor and regulatory T cells block the proliferation of self-reactive lymphoid cells, usually through the secretion of soluble factors. Upon recognition by a self-reactive precursor T cell, the self-reactive cells are suppressed. A network of antibodies and T cell receptors may develop the capability to react against the reactive components of self-reactive antibodies and T cells receptors, with the network of antibodies and T cell receptors also known as an anti-idiotype network. The antibodies and T cell receptors then neutralize self-reactive cells. Veto cells are T cells that express a self antigen.
The principal problems associated with organ transplantation are immune rejection and a shortage of acceptable donors. Unless the donor is an identical twin, the immune system of the recipient recognizes the graft as foreign and the recipient's immune system tries to reject the graft. Although immune suppression may postpone rejection for prolonged periods, immune suppression places the recipient at risk for infections and malignancies. Despite requiring chronic immune suppression, most organ and tissue transplants are successful in saving lives and improving the quality of life. The list of successfully transplanted tissues includes: kidney, heart, lung, liver, corneas, pancreas, pancreatic islets of Langerhans, intestines, brain tissue, liver, spleen, thymus, lymph nodes, bone marrow, skin, and bones. Combinations of tissue have also been transplanted; for example, heart-lung transplants, pancreas-kidney transplants, and pancreas-kidney-intestinal transplants.
Because of the relative success of the above organ and tissue transplants, a marked shortage of human organ donors exists. For example, although nearly 9,500 kidney transplants are performed annually in the United States, approximately 40,000 Americans develop end stage renal disease annually, and these 40,000 Americans could benefit from organ transplants. Xenografts, herein defined as transplants from another species, could potentially resolve the shortage of transplantable organs and tissues, but the risk of rejection is considered to be even greater than for allografts, herein defined as transplants from a non-identical donor of the same species.
Because of the severe shortage of human organ donors, transplant recipients have occasionally received a xenograft for short term life support, with the short term xenograft also referred to as a bridge transplant. By using bridge transplants of xenografts, additional time is provided to locate a suitable human donor.
Immune tolerance for new transplant grafts has been induced in graft recipients using bone marrow transplants. The patient's immune system is destroyed with high dose chemotherapy and/or total body irradiation. Autologous marrow is then infused into the patient simultaneous with the patient being exposed to the transplant or corresponding transplant antigens. As the immune system of the patient reconstitutes, the immune system recognizes the transplant antigens as self, along with the patient's own antigens. Although the procedure may be used for children and adults, the procedure exposes the patients to long term immune deficiency while the immune system repopulates and reconstitutes in the patient. Thus the patient is at considerable risk for infections and malignancies. Aggressive chemotherapy and/or irradiation may also be toxic to many of the patient's organs and tissues; for example, the lung, the liver, and the intestines.
The fetal or neonatal period offers a window of opportunity to develop tolerance to new antigens with less danger to the patient. When a fetus or neonate, i.e. newborn, is exposed to foreign antigens, including tissues from another species, the fetus or neonate, after maturation, is later specifically tolerant and capable of accepting grafts from the original source of the foreign antigen without immune suppression. Therefore, human fetuses may be exposed to antigens from a potential donor and receive post-natally a graft from the potential donor.
The donor is not limited to being human; i.e. other species may serve as donors for the human fetus exposed in the above manner. For example, certain congenital heart and hematological diseases may be diagnosed before birth using echocardiography or amniocentesis. A human fetus with a left ventricular syndrome may receive an intrauterine infusion of baboon cells from a specific donor baboon. After birth, the infant would receive a heart by transplant from the specific donor baboon using the above method. The infant, on receiving the transplanted heart, does not require immune suppression. Although the above method represents an elegant application of basic immunology principles, the method fails to address the transplant needs of the vast majority of patients having diseases recognized or diagnosed after birth, long after the prenatal or neonatal window of opportunity closes for the patients.
A variety of methods have been investigated for suppression of graft rejection, including gene therapy; transplants conducted on human fetuses by bone marrow replacement or other methods; and immune suppression by various drugs. Other studies focus on inducing immune tolerance to transplantation for allografts and xenografts by the introduction of foreign tissue in neonatal and post-natal recipients. The art discloses infusing human cells into non-human animals during the earliest stage of development of the non-human animal for incubating and harvesting hTCGF, and the art also discloses inducing transplantation immune tolerance by bone marrow transplantation or fetal stem cells to develop chimeras. Throughout, immune tolerance is induced within the transplant organ recipient.
U.S. Pat. No. 4,624,917 to Suaimoto discloses a process for producing human T-cell growth factor (hTCGF) by infusing human cells capable of producing hTCGF into non-human warm blooded animals, with the animals preferably at an immature stage; i.e. as eggs, embryos, fetuses, or newborn or infant animals. The development of the animal allows the infused human cells to develop and reproduce for later harvesting of the hTCGF.
U.S. Pat. No. 5,004,681 to Boyse et al. teaches obtaining hematopoietic stem cells and progenitor cells from neonatal or fetal blood. The obtained cells are cryogenically preserved for later use in hematopoietic or immune reconstitution and in gene therapy. U.S. Pat. No. 5,061,620 to Tsukamoto et al. teaches a method for isolating human hematopoietic stem cells in substantially homogeneous quantities.
Japanese Abstract No. 126519 teaches cultivating juvenile cells having the same genes and cytoplasm of an aged or sick person using an actual or artificial uterine environment, a normal cell incubator, or a cell propagation promoter. The cultivated juvenile cells are later used in parts of the body of the aged or sick person. Japanese Abstract No. 63-170322 teaches cultivating monogenetic cells for later transplanting to developed cells. Japanese Abstract No. 1-132528 discloses using the immune suppression property of human immunoglobulin GI protein (IgGl) by injection of IgGl into a recipient prior to transplantation. Japanese Abstract No. 63-39820 teaches using immune suppression drugs during transplantation of juvenile cells into an aged recipient.
H. Auchincloss, Jr., "Xenogeneic Transplantation--A Review", TRANSPLANTATION, Vol. 46, No. 1, July 1988, pp. 1-20, discusses the methods implemented for xenogeneic transplantation. In particular, Auchincloss discloses the induction in the recipient of neonatal tolerance for allografts and xenografts by the introduction of donor antigens in the recipient at the neonatal or embryonic stage of life. Auchincloss speculates toward achieving tolerance induction by using the principle of presenting foreign antigens at the time of maturation of T lymphocytes to enable the cells to consider the foreign antigens as self, thereby avoiding the development of the functions or suppressing the functions of the cells responsive to the foreign determinants.
R. E. Billingham et al., "Actively Acquired Tolerance of Foreign Cells", NATURE, Vol. 172, Oct. 3, 1953, pp. 603-606, discusses "actively acquired tolerance" to initiate tolerance by the first presentation of foreign tissue during the fetal phase, with resistance to a later transplanted grafts being abolished or reduced. M. Simonsen, "Artificial production of immunological tolerance. Induced tolerance to heterologous cells and induced susceptibility to virus", NATURE, Vol. 174, Apr. 30, 1955, pp. 763-764, demonstrates neonatal tolerance to xenoantigens (from a different species) by injecting the xenogeneic cells into bird embryos. The subsequent titers of natural antibodies to the donor were significantly reduced.
R. D. Owen, "Immunogenetic consequences of vascular anastomoses between bovine twins", SCIENCE, Vol. 102, 1949, pp. 400-401, suggests that the tolerance between dissimilar bovine littermates is due to exchange of blood during fetal development due to vascular connections within the placenta between the litter mates. J. W. Streilein, "Neonatal tolerance of H-2 alloantigens. Procuring graft acceptance the `old-fashioned` way", TRANSPLANTATION, Vol. 52, July 1991, pp. 1-10, reviews the mechanisms of neonatal tolerance. Depending on the combination of allogeneic murine strains, this can be due to negative selection, anergy, or suppression.
A. W. Flake et al., "In Utero Stem Cell Transplantation", EXPERIMENTAL HEMATOLOGY, Vol. 19, 1991, pp. 1061-4, discusses future directions for in utero transplantation of hematopoietic stem cells (HSC), including prenatal-specific tolerance induction for post-natal allogeneic and xenogeneic transplantation.
E. D. Zanjani et al., "Engraftment and Long-Term Expression of Human Hemopoietic Stem Cells in Sheep Following Transplantation In Utero", J. CLIN. INVEST., Vol. 89, April 1992, pp. 1178-1188, discusses inducing tolerance in sheep by transplanting hematopoietic stem cells from human fetal donors to sheep fetuses. The authors also teach the use of growth factors such as recombinant human IL-3 and GM-CSF to enhance donor hematopoiesis within the xenogeneic fetus. They suggest that hematopoietic stem cells can be stored and expanded as a "reservoir of human HSC" within the fetus for later use. They suggest that human immunoglobulins may also be produced in utero. E. D Zanjani et al., "Hematopoietic Chimerism in Sheep and Nonhuman Primates by In Utero Transplantation of Fetal Hematopoietic Stem Cells", BLOOD CELLS, Vol. 17, 1991, pp. 349-363, discloses transplanting fetal stem cells to an unrelated fetal animal, resulting in long-term stable hematopoietic chimerism.
E. F. Srour et al., "Sustained Human Hematopoiesis in Sheep Transplanted In Utero during Early Gestation with Fractionated Adult Human Bone Marrow Cells", BLOOD, Vol. 79, No. 6, 1992, pp. 1404-12, teaches the concept of fetuses representing the ideal host for HSC transplantation, and of transplanting human cells enriched for hematopoietic progenitor and stem cells to produce chimera. As with the previous articles (Zanjani et al), the studies are described as a preclinical study for treatment of human fetuses. Human fetuses would receive allogeneic cells in utero. C. Ezzell, "Sheep chimera makes human blood cells", SCIENCE NEWS, Vol. 141, Mar. 21, 1992, p. 182, comments on the Srour study (above) and lists human genetic blood disorders that can be diagnosed in utero and therefore treated by intrauterine infusion of cells.
M. Tavassoli et al., "Enhancement of the Grafting Efficiency of Transplanted Marrow Cells by Preincubation with Interleuidn-3 and Granulocyte-Macrophage Colony-Stimulating Factor", BLOOD, Vol. 77, April 1991, pp. 1599-1606, demonstrates that preincubation of murine bone marrow cells with IL-3 or GM-CSF enhanced subsequent engraftment in irradiated syngeneic hosts. B. W. Duncan et al., "Immunologic Evaluation of Hematopoietic Chimeric Rhesus Monkeys", TRANSPLANT. PROC., Vol. 23, February 1991, pp. 841-3, evaluates T cell maturation and function after fetal rhesus monkey hematopoietic cells from a fetal liver are injected into a mismatched rhesus fetus.
T. M. Crombleholme et al., "Transplantation of Fetal Cells", AM. J. OBSTET. GYNECOL, Vol. 164, January 1991, pp. 218-230, reviews the art for the use of fetal tissue donors and fetal tissue recipients. The review lists multiple human congenital hematologic diseases that could be treated by a marrow infusion when the patient is a fetus. Fetal tissue can also be used for donation, such as pancreatic islet cells for the treatment of diabetes mellitus and dopaminergic neurons for the treatment of Parkinson's disease. The art does not propose alterations of the fetal tissue prior to transplantation. C. G. Groth et al., "Evidence of xenograft function in a diabetic patient grafted with porcine fetal pancreas" TRANSPLANT. PROC., Vol. 24, June 1992, pp. 972-973, transplanted pancreatic islet cells from fetal pigs into a diabetic patient. The islets were not altered prior to transplantation.
The art concerning human-SCID mouse chimeras demonstrates that human lymphocytes can differentiate within a SCID mouse and become tolerant to the mouse. The human-SCD mouse chimera art teaches that tolerance to the mouse is by negative selection and possibly anergy, but not by suppression.
Mosier et al., "Transfer of a Functional Human Immune System to Mice with Severe Combined Immunodeficiency", NATURE, Vol. 335, 1988, pp. 256-259, discusses the expansion and differentiation of human lymphoid cells from peripheral blood within the SCID mouse. D. E. Mosier., "Immunodeficient Mice Xenografted with Human Lymphoid Cells: New Models for In Vivo Studies of Human Immunobiology and Infectious Diseases" J. CLIN. IMMUNOL. Vol. 10, 1990, pp. 185-191, and D. E. Mosier, "Adoptive Transfer of Human Lymphoid Cells to Severely Immunodeficient Mice: Models for Normal Human Immune Function, Autoimmunity, Lymphomagenesis, and AIDS", ADV. OL., Vol. 50, 1991, pp. 303-325 review the potential uses of these models such as the study of AIDS, the immune reactions to infectious diseases, and effect of drugs on the human immune system.
J. M. McCune et al., "The SCID-hu Mouse: Murine Model for the Analysis of Human Hematolymphoid Differentiation and Function", SCIENCE, Vol. 241, 1988, pp. 1632-1639, teaches the implantation of human fetal thymus, lymph node, or fetal liver hematopoietic stem cells in the SCID mouse and the differentiation of human lymphocytes and immunoglobulins. R. Namikawa et al., "Long-Term Human Hematopoiesis in the SCID-hu Mouse", J. EXP. MED., Vol. 172, October 1990, pp. 1055-1063, discloses co-implantation of human fetal thymus and liver in the SCID mouse leading to long term survival of human hematopoietic and lymphoid cells without the development of graft-vs-host disease.
B. Peault et al., "Lymphoid Reconstitution of the Human Fetal Thymus in SCID Mice with CD34+ Precursor Cells", J. EXP. MED., Vol. 174, November 1991, pp. 1283-1286, teaches that infusion of human hematopoietic stem cells leads to repopulation of human thymus fragments in the SCID mouse with human cells. J. F. Krowka et al., "Human T Cells in the SCID-hu Mouse are Phenotypically Normal and Functionally Competent", J. IMMUNOL., Vol. 146, June 1991, pp. 3751-3756, teaches that immature human cells mature into populations of mature T cells within SCID mice receiving fetal implants of human thymus and liver. B. A. E. Vandekerckhove et al., "Clonal Analysis of the Peripheral T Cell Compartment of the SCID-hu Mouse", J. IMMUNOL., Vol. 146, June 1991, pp. 4173-4179, teaches the maturation of human lymphocytes from fetal thymus and liver fragments into mature T cells with polyclonal and alloreactive T cell receptors, but without self-reactive T cell receptors.
H. Kaneshima et al., "Today's SCID-hu Mouse", NATURE, Vol. 348, December 1990, pp. 561-562, J. M. McCune et al., "The SCID-hu Mouse: Current Status and Potential Applications", CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY, Vol. 152, 1989, pp. 183-193, J. M. McCune, "SCID Mice as Immune System Models", CURRENT OPINION IN IMMUNOLOGY, Vol 3, 1991, pp. 224-229, and J. M. McCune, "The SCID-hu Mouse: a Small Animal Model for the Analysis of Human Hematolymphoid Differentiation and Function", BONE MARROW TRANSPLANTATION, Vol 9 (Suppl 1), 1992, pp. 74-76, review the SCID-hu model and possible applications in the study of human immunobiology, such as the study of AIDS and anti-HIV drugs, hematopoiesis, and immune reactions.
W. Huppes et al., "Acute Human vs. Mouse Graft-vs.-Host Disease in Normal and Immunodeficient Mice", EUR. J. IMMUNOL., Vol. 22, 1992, pp. 197-206, teaches that human peripheral lymphocytes can survive in mice that are artificially immunosuppressed or hereditarily immunodeficient such as the SCID mouse, providing natural antibodies are removed. Engraftment with large numbers of human lymphocytes, however, was associated with severe graft-vs-host disease (human vs. mouse).
M. Tary-Lehmann and A. Saxon, "Human Mature T cells that are Anergic In vivo Prevail in SCID Mice Reconstituted with Human Peripheral Blood", J. EXP. MED., Vol. 175, February 1992, pp. 503-516, teach that human cells mature into memory T cells with alpha/beta T cell receptors, but that the cells are anergic and fail to be stimulated by anti-CD3 antibodies. The anergy was reversible. T. Tscheming et al., "CD3+ T Cells in Severe Combined Immunodeficiency (SCID) mice. V. Allogeneic T Cells Engrafted into SCID Mice do not Induce Graft-versus-Host Disease in Spite of the Absence of Host Veto and Natural Suppressor Cell Activity", SCAND. J. IMMUNOL., Vol. 34, 1991, pp. 795-801, teaches that the tolerance of allogeneic murine lymphocytes engrafted within SCID mouse to the SCID mouse is not due to host veto cells or to natural suppressor cells. M-G. Roncarolo and B. Vandekerckhove, "SCID-hu Mice as a Model to Study Tolerance after Fetal Stem Cell Transplantation", "BONE MARROW TRANSPLANTATION", Vol. 9 (Suppl 1), February 1992, pp. 83-84, and B. A. E. Vandekerckhove et al., "Human Hematopoietic Cells and Thymic Epithelial Cells induce Tolerance via Different Mechanisms in the SCID-hu Mouse Thymus", J. EXP. MED., Vol. 175, April 1992, pp. 1033-1043, produce SCID-hu chimeras using a fetal thymus from one donor and fetal liver hematopoietic cells from another donor. The lymphocytes derived from the two human donors are tolerant to each other. Tolerance to the liver donor was by negative selection whereas tolerance to the thymus donor did not involve negative selection, but presumably by anergy. Most notably, mixing studies indicate that the immune tolerance was not due to suppression.
Y. J. Zeng et al., "Long-term Survival of Donor-Specific Pancreatic Islet Xenografts in Fully Xenogeneic Chimeras (WF Rat.fwdarw.B10 Mouse)", TRANSPLANTATION, Vol. 53, February 1992, pp. 277-283, teaches that mice treated with lethal irradiation and rat marrow cells were later tolerant to and accepted pancreatic islet cells from rats. S. T. Ildstad et al., "Cross-Species Transplantation Tolerance: Rat Bone Marrow-Derived Cells can Contribute to the Ligand for Negative Selection of Mouse T Cell Receptor V Beta in Chimeras Tolerant to Xenogeneic Antigens (Mouse+Rat.fwdarw.Mouse)", J. EXP. MED., Vol. 175, January 1992, pp. 147-155, discloses that when mice are lethally irradiated and transplanted with a mixture of rat and murine marrow cells, the chimera is tolerant to the corresponding rat and murine histocompatibility antigens. The tolerance is due to negative selection. A. M. Posselt et al., "Induction of Donor-Specific Unresponsiveness by Intrathymic Islet Transplantation", SCIENCE, 1991, pp. 1293-6, demonstrates the development of tolerance to pancreatic islets in allogeneic rats by injection of donor islets into the thymus after treatment with antilymphocyte serum. Tolerance is demonstrated to be due to negative selection rather than suppression.
K. Hamano et al., "The Effect of Intrathymic Injection of Donor Blood on the Graft versus Host Reaction and Cardiac Allograft Survival in the Rat", IMMUNOLOGY AND CELL BIOLOGY, Vol. 69, 1991, pp. 185-189, demonstrated a decreased graft-vs-host reaction when the donor of the hematopoietic and lymphoid cells received a previous intrathymic injection of host strain cells. Since the donor provided all of the immunoreactive cells and the host provided the target organs, this experiment is equivalent to injecting organ donor cells into the thymus of an organ recipient. Indeed, it was described as a model for heart graft rejection after injection of heart donor cells into the thymus of the heart graft recipient. Because the immune reactive cells of the host were killed by lethal irradiation, the mechanism of tolerance in the lymphocyte donor is irrelevant. Tolerance due only to negative selection or anergy could be transferred to the host as well as suppressor cells.
G. M. Williams et al., "Host Repopulation of Endothelium", TRANSPLANT. PROC., Vol. 3, March 1971, pp. 869-72 discloses that the endothelial cells of aorta grafts are replaced in 2 to 4 months by recipient endothelial cells. In bone marrow chimeras, there was partial repopulation of the endothelial cells in the bone marrow by donor derived cells. R. P. Gale et al., "Bone Marrow Origin of Hepatic Macrophages (Kupffer Cells) in Humans", SCIENCE, Vol. 201, September 1978, pp. 937-938, teaches that the Kupffer cells in the liver of allogeneic marrow recipients are replaced with donor cells.
The art demonstrates that transplant organ grafts can be partially repopulated with recipient cells within the organ graft recipient and that the marrow vascular endothelium in bone marrow recipients partially repopulate with donor cells.
D. Shafer et al., "Studies in Small Bowel Transplantation. Prevention of Graft-versus-Host Disease with Preservation of Allograft Function by Donor Pretreatment with Antilymphocyte Serum", TRANSPLANTATION, Vol. 45, February 1988, pp. 262-269, and D. Shafer et al., "Prevention of Graft-versus-host Disease Following Small Bowel Transplantation with Polyclonal and Monoclonal Antilymphocyte Serum", TRANSPLANTATION, Vol. 52, teaches that GvHD by lymphocytes from the intestine is a major problem after intestinal transplants. They disclose that GvHD can be prevented by treating the donor with antilymphocyte serum (ALS, either polyclonal or monoclonal) at the time of or before transplantation. The effectiveness correlates with the depletion of lymphocytes in the attached mesenteric lymph nodes. The graft is not repopulated ex vivo with organ graft recipient cells. Nor does the treatment with ALS remove the cellular targets of rejection, including endothelium, macrophages, dendritic cells, plasma cells, etc. Although the risk of GvHD from the intestinal transplant is reduced, the graft is still at risk for rejection and is still immune deficient and at risk for infections. The authors note that "its use in clinical transplantation may be limited by time or logistical constraints . . . "
G. E. Shafer et al., "Expression of a Swine Class II Gene in Murine Bone Marrow Hematopoietic Cells by Retroviral-Mediated Gene Transfer", Proc. Natl. Acad. Sci. USA, Vol. 88, November 1991, pp. 9760--9764, and D. W. Emery et al., "Expression of Allogeneic Class II cDNA in Swine Bone Marrow Cells Transduced with a Recombinant Retrovirus", TRANSPLANT. PROC., Vol. 24, April 1992, pp. 468-469, suggests that allogeneic and xenogeneic tolerance can be achieved by transgenic engineering and insert the genetic code for swine class II MHC antigens into murine myelopoietic precursor cells and into allogeneic swine cells. Tolerance to the organ donor would be induced in the organ recipient by inserting the donor MHC antigen genes into the recipient hematopoietic stem cells and performing an autologous bone marrow transplant on the recipient using the altered stem cells. The authors do not propose that the genetically altered cells replace the organ donor cells in the graft organ.
The relevant art also does not disclose a protocol for inducing transplantation immune tolerance in the organ donor animals prior to transplantation of grafts from the donor animals to the organ graft recipient. Nor does the prior art suggest the production and expansion of antigen specific suppressor cells, veto cells, cells producing anti-idiotype antibodies or anti-idiotypic antibodies responsible for immune tolerance within the organ graft donor for harvest and transfer back to the organ graft recipient.